The subject invention is directed to models for cell migration, and more particularly to in vitro and in vivo models which are especially useful for studying fibroblast cell migration during wound healing.
Throughout this application various publications are referenced, many in parenthesis. Full citations for each of these publications are provided at the end of the Detailed Description. The disclosures of each of these publications in their entireties are hereby incorporated by reference in this application.
After injury, a fibrin-rich clot fills the wound as a result of the local extravasation of plasma (Clark 1993b). This fibrin-rich provisional matrix, which also contains other matrix components like fibronectin (FN) and vitronectin as well as fibrin (Clark et al. 1981), forms a scaffold for the inward migration of the cells involved in early wound repair (Clark 1993a; Clark 1993b; Clark et al. 1982a; Clark 1996; Clark et al. 1996). Concomitantly, platelets release a plethora of growth factors, some of which bind to the fibrin meshwork. Subsequently, blood leukocytes, especially neutrophils and monocytes, migrate into the fibrin-rich provisional matrix. The neutrophils phagocytize and kill contaminating microorganisms (Tonnesen et al. 1988), while the monocytes mature into growth factor-producing macrophages (Shaw et al. 1990). Fibrin undergoes intermolecular crosslinking by formation of xcex5-(xcex3-glutamyl)lysine [xcex5-(xcex3-glu)lys] isopeptide bonds in the presence of plasma transglutaminase (Pisano et al. 1968). Crosslinking of xcex3 chains within fibrils forms dimers while intermolecular crosslinking among xcex1 chains creates oligomers and larger chain polymers (Mosesson et al. 1989; Shainoff et al. 1991). More recent in vitro studies indicate that in addition to xcex3 dimers, higher order forms of crosslinked xcex3 chain multimers form slowly and progressively over a period of hours to days (Siebenlist and Mosesson 1992). Coagulation factor XIIIa stabilizes the structure of the provisional matrix by cross-linking fibrin homocomplexes (Lorand 1972), and fibrin and FN heterocomplexes (Mosher 1975; Mosher 1976). FN is probably an important part of the invasion process in vivo since it is able to bind to cells and to other extracellular matrix (ECM) proteins simultaneously, and since fibroblasts can use FN as a substrate for migration in vitro (Hsieh and Chen 1983). Simultaneous with clot evolution during the first three days after injury, fibroblasts and endothelial cells in the underlying subcutaneous tissue proliferate (Clark 1993b). Fibroblasts and endothelial cells appear most numerous along the edge and base of the wound, with lesser numbers further away. By the third day after injury increased numbers of fibroblasts greatly expand the subcutaneous fibrous septae coursing between the fat lobules beneath the wound, and envelop individual adipocytes in proximity to the wound as well. Nevertheless, no fibroblast invasion of the wound clot is observed. Endothelial cells within blood vessels adjacent to the wound proliferate, causing marked vessel hypertrophy but do not move into the wound space (Clark et al. 1982b; Clark et al. 1982c).
Three days after injury, fibroblasts expressing abundant provisional matrix integrins (Xu and Clark 1996) migrate from the collagenous matrix of the dermis into the wound matrix as part of granulation tissue formation. Presumably this transmigration is in response to platelet-derived growth factor (PDGF) (Seppa et al. 1982; Senior et al. 1983) and other growth factors released by platelets and monocytes (Shimokado et al. 1985; Ross et al. 1986; Rappolee et al. 1988). Although PDGF is a potent mitogen and chemoattractant for fibroblasts (Seppa et al. 1982; Ross and Raines 1990), the full set of functional requirements for fibroblast transmigration from one matrix into another have not been defined.
Due to the complexity of the in vivo situation, three different in vitro models have been used in the past to study fibroblast invasion of a fibrin clot. Graham et al. (Graham et al. 1984) embedded explants of chick flexor tendons in a fibrin matrix and studied the migration, proliferation, and collagen synthesis of the fibroblasts. Migration in this system was induced by fetal bovine serum (FBS) but not by physiological concentrations of platelet lysate or PDGF. Brown and his coworkers (Brown et al. 1993) overlaid fibroblasts attached to tissue culture dishes with a thin fibrin layer and observed that cells migrated into the fibrin within 24 hours. This migration process was dependent on the nature of the fibrin clot: cross-linking of the fibrin xcex1-chains by factor XIIIa enhanced the number of invading cells. Knox et al. observed that the presence of FN (Knox et al. 1986) and plasminogen and its activators (Knox et al. 1987) were necessary for fibroblast invasion of a plasma clot on which the fibroblasts were seeded.
Before invasion of the fibrin clot, however, and in contrast to these in vitro models, resident tissue fibroblasts in normal dermis are surrounded by a matrix mainly composed of type I collagen. An often used in vitro model for the dermis is a relaxed hydrated collagen gel with embedded fibroblasts which acquire the dermal phenotypic characteristics of resident dermal fibroblasts (Elsdale and Bard 1972; Bell et al. 1979; Grinnell 1994). Just as in vivo, there is a low level of cell proliferation (Sarber et al. 1981) and collagen biosynthesis (Mauch et al. 1988), but an increased release of collagenase (Nusgens.et al. 19814; Unemori and Werb 1986). Furthermore, fibroblasts in relaxed collagen gels are less responsive to growth factors (Nakagawa et al. 1989; Nishiyama et al. 1990). An explanation for the different behavior of cells in the collagen gel in comparison to tissue culture plastic is that different kinds of extracellular matrix can dramatically affect cell functions and behavior by regulating gene expression and second messenger pathways (Hay 1991; Clark et al. 1995; Streuli et al. 1995; Tremble et al. 1995; Xu and Clark 1996). For example, when fibroblasts are cultured in collagen gels, xcex12 integrin gene expression is increased (Klein et al. 1991; Xu and Clark 1996) and the autophosphorylation of PDGF-receptor is decreased (Lin and Grinnell 1993).
Integrins are a family of cell surface receptors which are primarily responsible for cell adhesion (Hynes 1992). All integrins are composed of one xcex1 and one xcex2 subunit. A large number of integrins are responsible for the interaction of fibroblasts with the proteins of the provisional matrix. The integrins xcex13xcex21, xcex14xcex21, xcex15xcex21 and xcex1vxcex23 mediate adhesion of adult human dermal fibroblasts to FN (Elices et al. 1991; Gailit et al. 1993; Gailit and Clark 1996). xcex1vxcex23 is the only known fibroblast integrin that recognizes fibrinogen (Smith et al. 1990; Gailit and Clark 1996). When wound fibroblasts migrate into the fibrin/FN-rich clot, xcex13xcex21 and xcex15xcex21 fibronectin receptors, but not xcex12xcex21 collagen receptors, are dramatically up-regulated (Xu and Clark 1996). xcex12xcex21 collagen receptors increase on wound fibroblasts later at day 7 when collagen accumulates in the wound area (Welch et al. 1990; Clark et al. 1995).
A need continues to exist for an understanding of the regulation of interactions between cells and extracellular matrix and the functional mechanisms involved during wound repair.
This need is met by the development of models for cell migration, including an in vitro model and an in vivo model, according to the subject invention. The in vitro model for cell migration comprises a first extracellular matrix containing a cell (the cell which will migrate) and a second extracellular matrix in physical contact with the first extracellular matrix. The first extracellular matrix simulates a first natural environment in which the cell naturally resides, and the second extracellular matrix simulates a second natural environment into which the cell naturally migrates from the first natural environment.
The in vivo model according to the subject invention comprises an animal model having a naturally occurring first extracellular matrix containing a cell, and a second extracellular matrix in physical contact with the first extracellular matrix. The first and second extracellular matrices are generally as described above for the in vitro model, except that the first extracellular matrix is part of an animal model.
Having thus described the in vitro and in vivo models according to the subject invention, uses of the models are numerous. The primary uses are for screening substances for their effect on cell migration, and for screening extracellular matrices for their effect on cell migration.
In one particular embodiment, the invention provides a defined, easily modulated, reproducible in vitro model which simulates the transmigration process from a homeostatic collagenous extracellular matrix into a provisional extracellular matrix environment that resembles early cutaneous wounds. The dermal equivalent was supplied by fibroblasts embedded in a three-dimensional relaxed collagen gel (Bell et al. 1979). The dermal equivalent was attached to a fibrin-fibril coated dish and surrounded by a freshly-formed fibrin gel. The transmigration of fibroblasts from the opaque collagen gel into the transparent fibrin gel could be quantified visually by counting the cells in the fibrin gel under an inverted microscope. This system allowed the study of the interactions between migrating cells, growth factors, and the physiological extracellular matrices present in this model of an early wound and the definition of the involved mechanisms and integrins. Fibroblast invasion of the fibrin gel was stimulated by platelet releasate, a natural mixture of growth factors, and by recombinant PDGF-BB, a cytokine found in vivo in the early phase of wound repair (Clark 1996). The migration was dependent on the presence of plasminogen. FN had to be present in both the collagenous and the provisional extracellular matrix to allow the transmigration process. Furthermore, the process was inhibited by RGD peptide and appeared to be mediated by xcex15xcex21 integrins.